Evaluation of FOXCUT, CCAT2, and HULC LncRNA Expression Levels and Apoptosis Induction by Sodium Butyrate in PC-3 and LNCAP Prostate Cancer Cell Lines

Sodium butyrate (NaBu) is a short-chain fatty acid acting as a histone deacetylase inhibitor, and has been shown to be a potential regulator of cancer cell death. This study aimed to evaluate the effect of NaBu on cell cycle control, apoptosis, and expression of some lncRNAs in two human prostate cancer cells (PC-3 and LNCAP). Cell viability was assessed and the appropriate dose was determined using the MTT assay. Real-time PCR technique was also used to evaluate the expression levels of HULC, FOXCUT, and CCAT2 lncRNAs. Apoptosis was diagnosed using annexin V staining, and cell cycle distribution was then assessed using flow cytometry with propidium iodide DNA staining. NaBu induced apoptosis in both prostate cancer cell lines in a dose-dependent manner. The expressions of CCAT2 and HULC lncRNAs genes have significantly decreased in the presence of NaBu (P <0.05) in both PC3 and LNCAP cell lines, in comparison with the control. However, no significant difference was observed in the expression of FOXCUT lncRNAs. Moreover, the results of flow cytometry showed an increase in cell cycle arrest of LNCAP cell line at the sub-G1 stage as compared to the control cells, but no significant difference was observed between the control cells and NaBu-exposed PC-3 cells. In addition, the percentages of early and late apoptotic cells following treatment with NaBu were 80% and 49.63% in LNCAP and PC-3 cells, respectively. Our results suggest that NaBu has a positive effect on the induction of apoptosis and inhibition of cell cycle in PC-3 and LNCAP prostate cancer cells.

The advanced prostate cancer is still incurable, and treatment for these patients is only an average survival of 2 years with no progression (2,3). Therefore, finding new drug therapies is very important in this regard. Sodium butyrate (NaBu) is a short-chain fatty acid that competitively binds to zinc sites of classes 1 and 2 histone deacetylases (HDACs). Accordingly, this binding can consequently affect histone acetylation, which ultimately creates a modified DNA structure that alters chromatin. Therefore, it enhances the chromatin's ability to access transcriptional regulatory complexes, leading to the increased transcriptional activation of various genes. Butyrate is a HDAC inhibitor, which stops the cell cycle at the G1 or G2/M stages, and increases the expressions of other genes and proteins involved in cell differentiation and apoptotic signaling (4)(5)(6). Long non-c0ding RNAs (lncRNAs) have a size range from about 200 nucleotides to less than 100 kb (7). It is possible that NaBu is not only a prophylactic agent in the large intestine of people that typically consumes high-fiber diets, but it may also act as a therapeutic agent (11)(12)(13). In recent years, changes in gene expression have been extensively studied in people with cancer. In this study, the transcriptional expression levels of three lncRNAs namely colon cancer associated transcript 2 (CCAT2), HULC, and FOXCUT, and the effect of NaBu on cell cycle control, apoptosis, and expressions of these lncRNAs were evaluated in two prostate cancer cell lines, PC-3 and LNCAP.

×100
The concentration of IC50 was estimated using Prism graph pad 6 software version 25 (14).

Detection of apoptotic cells by flow cytometry
Cell apoptosis was assessed by annexin V staining kit (IQ Product, Germany) followed by the flow cytometry analysis. The PC-3 and LNCAP cell lines were poured into 6 cell plates for 24 h; 2.5 mM NaBu was then added, and after 48 h the cells were trypsinized, centrifuged, and afterwards, annexin V was incubated for 15 min at room temperature for evaluation by flow cytometry (15).  Table 1. Finally, the relative expression level of the genes was calculated using 2 -∆∆ct method.

Investigation of cell cycle inhibition
Firstly, the PC-3 and LNCAP cells (500,000 cells per well) were poured into each well in 6 plates, and after 24 h the supernatant culture medium of the cells was completely emptied, treated with fresh whole culture medium containing 2.5 mM NaBu, and then incubated for 5 h.
Subsequently, the cells were washed with PBS and trypsin, isolated from the bottom of the plate and after re-washing with PBS, they were prepared for flow cytometry (16). Distribution in different phases of the cell cycle called sub-G1, G0 / G1, G2 / M, and S was also examined.

Statistical analysis
Statistical significance between treatment and control groups were obtained using two-way analysis of variance and the Turkey's tests (SPSS Statistics software, version 22). Data were presented as the mean ± standard deviation of three replicates from three independent experiments.

Investigation of cell viability based on colorimetric MTT assay
In this study, MTT method was used to  Table 1. Sequence of primers for GAPDH, HULC, FOXCUT, and CCAT2 lncRNAs genes. group (P <0.05). At 1 mM, there was a significant difference with the control group after 24 and 72 h of exposure, while this difference was not significant after 48 h ( Figure 1). NaBu-exposed PC-3 cells for all the time intervals at 0.5 mM concentration did not significantly increase the cytotoxicity in comparison with the control groups ( Figure 1A). Figure 1B indicated the cell viability of LNCAP that revealed a significant cytotoxic activity at any concentration after 72 h exposure to NaBu. Comparison of the means in cell lines were also performed using the two-way ANOVA.

Results of gene expression using qRT-PCR
In the present study, the quantitative real time of sub-G1 population in NaBu-exposed PC-3 cells; however, no significant change was observed in the treated group as compared to the control group.

Apoptosis evaluation by flow cytometry
Annexin V is used in flow cytometry to detect apoptotic cells through its ability to bind to phosphatidyl serine, which is known as a marker of   (10). FOXC1 also acts as a downstream target for growth factorconverting β (TGF-β) to prevent tumorigenesis.
Increased FOXC1 expression causes cells to be sensitive to TGF-β1-mediated growth inhibitory effects and stop cells in the G0/G1 phase (18).
Here, we evaluated the up regulation of FOXCUT in LNCAP cells. However, we have observed no difference between the control and treatment groups in terms of the FOXCUT lncRNA expression. In